The use of herbicides in weed control is a widely accepted agricultural practice. Our understanding of herbicide metabolism and degradation is still in its infancy and is being actively investigated. Soil microorganisms were implicated in the degradation of herbicides by Joshi et al., Weed Sci. 33: 888-893, 1985. Sulfonylurea herbicides were shown to be co-metabolized by the soil bacterium Streptomyces griseolus by Romesser et al., Abstr. Ann. Mtg. Am. Soc. Microbiol. p. 248, 1985. Further study, as disclosed by Leto et al., Plant Physiol. 805: 5347 (1986) and Romesser et al., Biochem. Biophys. Res. Comm. 140: 650-659 (1986) showed that two cytochrome P450 enzymes designated P450SU1 and P450SU2, heme containing proteins of about 45,000 molecular weight, are synthesized in cells of the bacterium Streptomyces griseolus when they are grown in a medium containing any of several herbicides. The synthesis of these proteins by S. griseolus is detectable by UV/vis difference spectroscopy as described by Romesser, et al., Biochem. Biophys. Res. Comm. 140: 650-659 (1986), analytical anion exchange and gel filtration chromatography as described by O'Keefe et al., Plant Physiol. 805: 5347 (1986) and LDS gel electrophoresis as described by Leto et al., Plant Physiol. 805: 5348 (1986). Romesser et al., Biochem. Biophys. Res. Comm. 140: 650-659 (1986) and O'Keefe et al., Recent Advances in Phytochemistry 21: 151-173 (1987), correlated the presence of P450 enzymes with the ability of this organism to carry out a variety of metabolic reactions on a number of sulfonylurea herbicides. Further, as discussed by Romesser et al., Biochem. Biophys. Res. Comm. 140: 650-659, 1986, crude cell-free extracts from S. griseolus exhibit sulfometuron methyl (10010) hydroxylase activity only when they are from cells grown in the presence of certain sulfonylureas, and difference spectra of the extracts resulting when chlorsulfuron (10013) or sulfometuron methyl (10010) is added suggest that the newly appearing cytochromes P450 bind to these compounds in a manner similar to substrate binding to cytochrome P450.
Additionally, genes that cause the breakdown of the active moieties of herbicidal compounds may be incorporated in plants and cause said plants to become resistant to the affected herbicide. Stalker et al., Science 242: 419-422 (1988) describe the transfer of the gene from Klebsiella ozaenae encoding a specific nitrilase that converts the herbicide bromoxynil to metabolite 3,5-dibromo-4-hydroxybenzoic acid into tobacco plants with the result that the tobacco plants became resistant to bromoxynil.
The major objects of the invention described here are the DNA sequences encoding the two cytochromes P450. Other objects are the sequences encoding their iron-sulfur protein electron donors. These sequences of this invention are from the bacterium Streptomyces griseolus ATCC11796. These two cytochromes P450 are capable of metabolizing sulfonylurea compounds and other herbicides. The two cytochromes P450 have been designated P450SU1 and P450SU2, and the two iron-sulfur proteins have been designated FeS-A and FeS-B.
In wild type Streptomyces griseolus, expression of cytochromes P450SU1, P450SU2, and iron-sulfur proteins FeS-A and FeS-B is induced by the addition of sulfonylurea compounds. Although many sulfonylurea compounds may be metabolized by these cytochromes P450, not all are good inducers of these proteins. Thus optimal metabolism of many sulfonylurea compounds by wild type organisms can only be achieved by first inducing the cytochromes P450 and iron-sulfur proteins with a sulfonylurea known to be a good inducer. Organisms producing the P450 enzymes constitutively or as a result of exposure to light would obviate the need for inducing organisms with sulfonylureas to make them capable of metabolizing said sulfonylureas.
Thus, another object of this invention is to obviate the need to induce the herbicide metabolizing cytochromes P450 and their iron-sulfur protein electron donors in organisms (bacteria and plants) by transforming said organisms with the genes of the herbicide metabolizing cytochromes P450 and where necessary, their iron-sulfur protein electron donors contained in plasmids which permit the constitutive or light induced expression of the P450 enzymes and, where necessary, the iron sulfur proteins in the transformed organisms. Said transformed organisms are able to metabolize herbicides, both good and poor inducers, whenever they encounter them.
Typical cytochrome P-450 monooygenase systems from bacteria are similar to the P-450 CAM system from Pseudomonas putida (Sligar et al. in: Cytochrome P-450 Structure, Mechanism and Biochemistry, Ortiz de Montellano, ed. Plenum Press, N.Y. (1986) pp. 429-504). This system is comprised of a flavoprotein reductase (putidaredoxin reductase), a low molecular weight iron-sulfur protein (putidaredoxin) and the cytochrome P-450 (P-450 CAM). This system of proteins functions to transfer reducing equivalents from a reduced pyridine nucleotide sequentially from putidaredoxin reductase, to putidaredoxin and then to P-450 CAM. It is important to note, however, that the specificity of the enzyme system for substrate resides solely on the P-450 protein, and that the reductase and iron sulfur proteins are only important insofar as they provide the reducing equivalents to the P-450 necessary for catalysis. Thus, another object of this invention is to place the genes for sulfonylurea or herbicide metabolism in other organisms in such a way as to utilize existing sources of reducing equivalents in these organisms to facilitate the function of the cytochrome P-450.